Project Summary ? Overall Advances in high-throughput phenotyping and molecular characterization technologies have had profound impacts on biological research. However, it is frequently difficult, for example, to study the functional consequences of genetic changes because of the lack of suitable analytical technologies to define the functional cellular state. Mass spectrometry (MS) is presently the most powerful approach for quantifying the molecular changes that arises from altered gene expression, but limitations have prevented it from being a routine and broadly available tool. Specifically, the sensitivity, throughput and precision of proteomics measurements are significantly less than other high-throughput technologies that are driving major advances in modern biology (e.g. sequencing and high-content imaging). In this renewal the Proteomics Resource has the goal of broadly impacting biomedical research by providing the abilities to: obtain high quality data from at least 10- to 100- fold smaller samples, produce much more comprehensive quantitative measurements, improve coverage of low abundance components such as problematic peptide isomers and post translational modifications, and enable the analysis of far larger sample sets than presently practical by providing large increases in measurement throughput. We will address the key deficiencies of proteomics using new Structures for Lossless Ion Manipulations (SLIM) ion mobility (IM)-MS based technologies that will vastly increase proteomic sample throughput as well as provide improved reproducibility, sensitivity, accuracy of quantification and coverage of proteomics measurements. These advances will be complemented by innovative approaches for rapid nanoliter scale robotic processing of much smaller samples than presently feasible, and include analyses of important functional `sub-proteomes' (e.g. phosphoproteomics and activity-based proteomics samples). The robotic sample processing will provide throughput matching the speed of the SLIM IM-MS platform. These advances will occur in conjunction with algorithmic and software developments needed to handle the SLIM IM-MS platform data flow and bioinformatics for obtaining biological insights from these data. In combination, our work will lead to the rapid implementation of the new capabilities through their application to a set of challenging biomedical projects. The developments will be disseminated to the broader community by both conventional (e.g. workshops and training activities) and direct approaches that include working directly to `seed' the new technology in a number of outside research laboratories with expertise in developing and implementing biomedical technologies, and also helping to facilitate commercial implementations from a number of vendors spanning multiple MS platform types. Our project will result in broad and impactful initial applications of advanced proteomics capabilities, and effective dissemination of these technologies by the end of this final renewal of the Resource.